The present invention relates to a DNA chain which provides higher carotenoid content during biosynthesis of carotenoid and a method for producing carotenoids characterized by introducing said DNA chain into carotenoid producing microorganism to express said chain and to obtain higher carotenoid content.
Carotenoid is a general name of a kind of natural pigments. Generally, carotenoids have 40 carbon atoms and consists of isoprene skeletons, and Carotenoids are abundant in the natural world. Approximately 600 kinds of carotenoids have been isolated and identified up to the present [(see Key to carotenoids. Basel-Boston, Birkhauser, 1987(Pfander, H. ed.)]. Carotenoids are synthesized through the isoprenoid biosynthetic pathway, a part of which is common to the pathways for steroids and other terpenoids. Passing through the isoprene common biosynthetic pathway, hydroxymethylglutaryl-CoA(HMG-CoA) is converted to isopentenyl pyrophosphate(IPP), which has 5 carbon atoms, via mevalonate. Then IPP is converted to dimethylallyl pyrophosphate(DMAPP) by isomerization. Then, by polycondensation with IPP which has 5 carbon atoms, DMAPP is converted sequentially to geranyl pyrophosphate(GPP which has 10 carbon atoms), farnesyl pyrophosphate(FPP which has 15 carbon atoms), geranylgeranyl pyrophosphate(GGPP which has 20 carbon atoms) and so forth (FIG. 1).
The carotenoid biosynthetic pathway is branched from the isoprene common pathway at the point of GGPP is formed. At the point, two molecules of GGPP are condensed to synthesize phytoene which is the first carotenoid and colorless. Then, phytoene is converted to lycopene by desaturation reaction. Then, lycopene is converted to xcex2-carotene by cyclization. Various xanthophylls such as zeaxanthin and astaxanthin are synthesized by introducing hydroxyl groups or keto groups to xcex2-carotene.
Recently, the inventors of the present invention cloned the carotenoid biosynthesis genes derived from Erwinia uredovora, which is a non-photosynthetic epiphytic bacterium in Escherichia coli by using yellowish color of Er. uredovora as markers and elucidated the functions of the genes. Then, various combinations of these genes are introduced to express, and it made possible that microorganisms such as E. coli and yeast produce phytoene, lycopene, xcex2-carotene, zeaxanthin and so forth(See FIG. 2): [See Misawa, N., Nakagawa, M., Kobayashi, K., Yamano, S., Izawa, Y., Nakamura, K. and Harashima, K., xe2x80x9cElucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products expressed in Escherichia colixe2x80x9d, J. Bacteriol., 172: 6704-6712 (1990); Misawa, N., Yamano, S., and Ikenaga, H., xe2x80x9cProduction of xcex2-carotene in Zymomonas mobilis and Agrobacterium tumefaciens by introduction of the biosynthesis genes from Erwinia uredovoraxe2x80x9d, Appl. Environ. Microbiol., 57: 1847-1849 (1991); Yamano, S., Ishii, T., Nakagawa, M., Ikenaga, H., and Misawa, N., xe2x80x9cMetabolic engineering for production of xcex2-carotene and lycopene in Saccharomyces cerevisiaexe2x80x9d, Biosci. Biotech. Biochem., 58: 1112-1114 (1994) and Japanese Patent Application laid-open No. HEI 3-58786(Japanese Patent Application filing No. HEI 2-53255):xe2x80x9cA DNA chain useful for synthesis of carotenoidsxe2x80x9d by the inventors of the present invention]. With the carotenoid biosynthesis genes from Er. uredovora, carotenoids can be synthesized from FPP. Since FPP is the common substrate not only for carotenoids but also for steroids and other terpenoids, bacteria incapable of synthesizing carotenoids also have FPP. Accordingly, for example, when four crt genes, crtE, crtB, crtI and crtY, which are necessary for biosynthesis of xcex2-carotene from FPP are introduced in microorganisms, the microorganism becomes capable of producing xcex2-carotene (See FIG. 2). Furthermore, by the same procedures as mentioned above, the inventors cloned the carotenoid biosynthesis genes derived from a marine bacterium, Agrobacterium aurantiacum in E. coli. By expressing various combinations of the genes from the bacterium and those from the above-mentioned Er. uredovora, it made possible that the microorganisms such as E. coli produce astaxanthin, canthaxanthin and so forth (See FIG. 3): (Norihiko Misawa et al., xe2x80x9cElucidation of an astaxanthin biosynthetic pathway at the level of the biosynthesis genesxe2x80x9d, Abstract of the 36th Symposium on the chemistry of natural products: 175-180 (1994)). Among the above carotenoids, astaxanthin, zeaxanthin and xcex2-carotene are already in practical use and are regarded as promising substances. They are used for food or feed additives as red or yellow natural coloring agents or as nutritional aid having cancer prophylactic activity, immunopotentiating activity or provitamin A activity. Accordingly, when the carotenoid biosynthesis genes obtained by the inventors is used as exogenous genes for transforming microorganisms such as E. coli to express, it gave microorganisms such as E. coli the capability of biosynthesis for producing useful carotenoids. Up to now, it is the only way to improve production of useful carotenoids was to find out microorganism which can synthesize sufficient amount of a targeted carotenoid, and to try to increase its production by investigating culture conditions or mutation treatment. Owing to the studies done by the inventors, it became possible to choose host microorganism which is cultured easily and proliferates rapidly, and is guaranteed to be safe for food regardless of its carotenoid producing capability. As a matter of course, it is also possible to use microorganisms which can synthesize sufficient amount of useful carotenoids originally. In such a case, by transforming the microorganisms with carotenoid biosynthesis genes, it became possible to obtain higher carotenoid production or to alter final carotenoid products. For example, when both crtW and crtZ genes from Ag. aurantiacum were introduced into a microorganism capable of producing xcex2-carotene as a final product to express them, the microorganism was transformed to another one which produce astaxanthin as a final product.
On the other hand, both astaxanthin and xcex2-carotene can also be synthesized by organic synthesis methods. In these cases, considering these carotenoids are used for feed or food additives, there is problems that by-products are also produced and such synthetic products are not preferred by consumers because they prefer natural products. However, carotenoids produced by the conventional fermentation methods could not compete with those by the organic synthesis methods in price. As mentioned earlier, when the above mentioned carotenoid biosynthesis genes are used, it improves the fermentation methods, thereby it is considered that the carotenoid produced by the fermentation methods will be able to compete with those by the organic synthesis methods in price. If the microorganism can accumulate enough amount of carotenoid in itself, the carotenoid produced by the microorganisms will succeed in such price competition. Therefore, a technology to obtain higher carotenoid content by using microorganisms has been longed for.
Until now, in order to obtain higher carotenoid production in its biosynthesis; the traditional random mutation method is only employed to select mutant strains having higher carotenoid content with mutagenic agent such as NTG. However, this method requires huge amount of time and labor of technicians. In addition, even if enhancement of carotenoid synthesis is successfully achieved, the method requires both huge amount of time and effort to inhibit decreasing of carotenoid content caused by frequent reverse mutations naturally happens because the method lacks its theoretical basis.
The object of the present invention is to increase amount of carotenoids biosynthetically produced by microorganisms.
To solve the above problem, the inventors have investigated the problem thoroughly and developed a novel technology which provides several times higher carotenoid production amount by introducing a DNA chain containing only one gene into a carotenoid producing microorganism to express the gene in them.
More specifically, the inventors of the present invention found the followings and completed the present invention. When a DNA chain containing a gene substantially encoding an amino acid sequence of IPP isomerase which converts IPP into DMAPP, is introduced in microorganisms such as E. coli having carotenoid synthesis gene derived from Er. uredovora and so forth, content of carotenoid in cells such as lycopene and xcex2-carotene becomes 1.5-4.5 times higher than that in control cells can be achieved. The gene substantially encoding IPP isomerase amino acid sequence which converts IPP into DMAPP was obtained from the astaxanthin producing microorganisms such as Phaffia rhodozyma and Haematococcus pluvialis. 
The characteristics of the DNA chain of the present invention are as follows.
(1) A DNA chain capable of increasing carotenoid production amount and containing the nucleotide sequence which encodes the polypeptide having the amino acid sequence substantially shown in SEQ ID NO: 1, or a DNA chain that can be hybridized with said DNA chain.
(2) A DNA chain capable of increasing carotenoid production and containing the nucleotide sequence which encodes the polypeptide having the amino acid sequence substantially shown in SEQ ID NO:2, or a DNA chain that can be hybridized with said DNA chain.
The present invention also relates to a method for carotenoid production. The characteristics of the carotenoids production methods of the present invention are as follows.
(3) A production method characterized by introducing the DNA chain mentioned above either (1) or (2) into carotenoid producing microorganism, culturing said transformed microorganism and increasing carotenoid content in the cells and culture broth.
(4) A production method characterized by introducing the DNA chain containing the nucleotide sequence which encodes the polypeptide having the substantially same amino acid sequence shown in SEQ ID NO:3, or a DNA chain that can be hybridized with said DNA chain into carotenoid producing microorganism, culturing said microorganism and increasing carotenoid content in the cells and culture broth.
The present invention is described herein below.
As described in before, by introducing the carotenoid biosynthesis gene derived from microorganisms such as Erwinia uredovora, the non-photosynthetic soil bacteria and Agrobacterium aurantiacum, the marine bacteria) into other microorganisms which do not produce carotenoids such as E. coli, the microorganism can produce useful carotenoids such as astaxanthin, zeaxanthin, xcex2-carotene and lycopene. In order to compete in price of the carotenoid produced by using the organic synthesis methods, it is necessary to achieve as higher carotenoid production as possible. The IPP isomerase gene, which include the gene encoding the polypeptide whose amino acid sequence is substantially IPP isomerase, of the invention is extremely useful for increasing the production amount of carotenoids. By using modern biotechnology, it is relatively easy to increase production amount of a protein encoded by an exogenous gene by enhancing expression level of the gene. However, if amounts of substrate necessary for a protein, that is enzyme, is limited, higher production of the protein does not lead to higher production of biochemicals such as carotenoids. For example, without sufficient amount of FPP, which is the first substrate, enhancement of expression level of the carotenoid synthesis genes does not lead to higher amount of carotenoids production. This time, we succeeded in increasing carotenoid production amount by introducing the IPP isomerase gene. It is considered that the introduction of the IPP isomerase gene makes the flow of, the upstream of the pathway up to FPP larger(more efficient) and consequently, increased supply of FPP led to higher carotenoid production amount. The present invention started from the findings that by introducing either the gene encoding IPP isomerase, which convert from IPP to DMAPP vise versa, or encoding the protein homologous to IPP isomerase into carotenoid producing microorganism such as E. coli, to express the gene, carotenoid production amount is increased. By using carotenoid biosynthesis genes from Er. uredovora, cDNA expression libraries of Phaffia rhodozyma, Haematococcus pluvialis and so forth were prepared in xcex2-carotene producing E. coli as a host. As increased xcex2-carotene content in E. coli made, some of the yellowish colonies brighter till almost orange. The plasmids extracted from such E. coli colonies were analyzed and were found to have genes with high homology to IPP isomerase of Saccharomyces cerevisiae. It has been speculated that HMG-CoA reductase(FIG. 1), which catalyzes the reaction from HMG-CoA to mevalonate, may be the rate limiting enzyme for terpenoids including carotenoids. However, as for IPP isomerase, any such report has not been presented. Therefore, increase of carotenoid production by introducing a IPP isomerase gene was a new finding.
The present invention provides a DNA chain having characteristics of increasing carotenoid production amount, and it containing the nucleotide sequence which encodes the polypeptide having the substantially same amino aid sequence as those of IPP isomerase, and a production method for carotenoid characterized by introducing said DNA chain into the carotenoid producing microorganism, culturing said transformed microorganism and increasing carotenoid content in the culture broth and cells.
The DNA chains of the present invention includes the DNA chains mentioned above (1) or (2), or the DNA chains which hybridize to said chains under stringent conditions.
Substantially, the polypeptides encoded by the DNA chains of the present invention have the amino acid sequences shown in SEQ ID NO:1 (A-B in FIGS. 4 and 5) or in SEQ ID NO:2 (C-D, in FIGS. 6 and 7). In the present invention, the polypeptides encoded by these DNA chains, the proteins of which amino acid sequence is substantially IPP isomerase, may be altered by deletion, replacement, addition and so forth of some amino acids, as long as the resulted polypeptides hold their higher carotenoid production activity. This allowance corresponds to xe2x80x9chaving the substantially same amino acid sequence substantially shown in SEQ ID NO:1 or SEQ ID NO:2xe2x80x9d. As an example, a sequence which lacks the first amino acid(Met) can be included as the altered polypeptide or the altered enzyme. Needless to say, the DNA chains of the present invention include not only the chains having the nucleotide sequences which encode the amino acid sequences shown in SEQ ID NOS 1 and 2 (FIGS. 4 to 5), but also the degenerate isomers of the chains, which differs only on degenerate codons and encode the same polypeptides as the original chains do.
(1) Obtaining the DNA Chains
One method to obtain a DNA chain having the nucleotide sequence which encodes the amino acid sequence of the above protein is chemical synthesis of the DNA chain at least a part of the chain according to the known nucleic acid synthesis method. However, considering that there are so many amino acids bound in the protein, it would be more preferable than chemical synthesis to make cDNA libraries of Haematococcus pluvialis or Phaffia rhodozyma or the like to obtain a targeted DNA chain by applying some popular method in the field of genetic engineering such as hybridization with appropriate probes.
(2) Transformation of Microorganisms Such As E. coli and Expression of Gene
Higher carotenoid content in culture broth or cells of microorganisms can be achieved by introducing the above mentioned DNA chain of the present invention into appropriate microorganisms such as carotenoid-producing bacteria such as E. coli and Zymomonas mobilis containing carotenoid biosynthesis genes from Erwinia uredovora and so forth, or carotenoid-producing yeast such as Saccharomyces cerevisiae containing carotenoid biosynthesis genes from Erwinia uredovora and so force.
The outline of the method to introduce exogenous genes into preferable microorganisms is mentioned below.
Procedures or methods to introduce and express exogenous genes in microorganisms such as E. coli, besides those mentioned below in the present invention, includes those widely used in the field of genetic engineering. Those are applicable to the invention. See xe2x80x9cVectors for cloning genesxe2x80x9d, Methods in Enzymology, 216: 469-631 (1992), Academic Press; xe2x80x9cOther bacterial systemsxe2x80x9d, Methods in Enzymology, 204: 305-636 (1991) Academic Press).
[E. coli]
There are some established and efficient methods to introduce exogenous genes to E. coli such as Hanahan""s method and rubidium method, and they are applicable to the present invention (See Sambrook, J., Fritsch, E. F., Maniatis, T., xe2x80x9cMolecular cloningxe2x80x94A laboratory manualxe2x80x9d, Cold Spring Harbor Laboratory Press (1989)). Expression of exogenous genes in E. coli can be performed by known methods (See xe2x80x9cMolecular cloningxe2x80x94A laboratory manualxe2x80x9d, ibid.), for example, vectors for E. coli such as pUC and pBluescript vectors having lac promoter can be used. The inventors of the present invention used pSPORTl vector or pBluescript II KS vector having lac promoter as vectors for E. coli, and inserted the IPP isomerase gene, derived from Haematococcus pluvialis, Phaffia rhodozyma or Saccharomyces cerevisiae, into the lac promoter with the direction of reading through of the transcription, and expressed the gene in E. coli. 
[Yeast]
There are some established methods such as the lithium method to introduce exogenous genes into Saccharomyces cerevisiae, yeast, and such methods are applicable to the present invention (See xe2x80x9cNew biotechnology on yeastxe2x80x9d, Ed. Bio-industry Association(Yuichi Akiyama, editor in chief), Igaku Syuppan Center). Expression of exogenous genes in yeast can be performed as follows. Using both promoters and terminators, e.g. for PGK and GPD, an expression cassette is constructed by inserting the exogenous gene so that during transcription, the gene is to be read through at the position between the promoter and the terminator. Expression can be performed by inserting the expression cassette into a vector for S. cerevisiae such as YRp vectors (multi-copy vectors for yeast, replication starts at ARS sequence of yeast chromosome), YEp vectors (multi-copy vectors for yeast, replication starts at 2 xcexcm DNA) and YIp vectors (vectors for yeast chromosome, no starting point of replication in yeast) (See xe2x80x9cNew biotechnology on yeastxe2x80x9d, ibid.; xe2x80x9cGenetic engineering for production of substancesxe2x80x9d, Ed. Japanese Society of Agrocultural Chemistry, Asakura Publishing company; or Yamano, S., Ishii, T., Nakagawa, M., Ikenaga, H., Misawa, N., xe2x80x9cMetabolic engineering for production of xcex2-carotene and lycopene in Saccharomyces cerevisiaexe2x80x9d, Biosci. Biotech, Biochem., 58: 1112-1114 (1994)).
[Zymomonas mobilis]
Introduction of exogenous genes into Zymomonas mobilis, the ethanol-producing bacterium can be performed by conjugal transfer method which is commonly used for gram negative bacteria. Expression of exogenous gene in Zymomonas mobilis can be performed by using pZA22 vector for this bacterium (See Katsumi Nakamura, xe2x80x9cMolecular breeding of Zymomonas bacteriaxe2x80x9d, Journal of the Japanese Society of Agrocultural Chemistry, 63: 1016-1018 (1989); and Misawa, N., Yamano, S., Ikenaga, H., xe2x80x9cProduction of xcex2-carotene in Zymomonas mobilis and Agrobacterium tumefaciens by introduction of the biosynthesis genes from Erwinia uredovoraxe2x80x9d, Appl. Environ. Microbiol., 57: 1847-1849 (1991)).
(3) Method to Increase Carotenoid Production in Microorganisms
By applying the above mentioned procedures or methods for introduction and expression of exogenous genes in microorganisms, both the carotenoid synthesis genes and the IPP isomerase gene can be introduced to express, and microorganisms capable of producing large amount of carotenoid can be obtained.
Farnesyl pyrophosphate (FPP) is the common substrate not only for carotenoids but also for other terpenoids such as sesquiterpenes, triterpenes, sterols and hopanols. In general, since microorganisms are synthesizing terpenoids even though they are not capable of synthesizing carotenoids, basically all of the microorganisms possesses FPP as an intermediate metabolite. On the other hand, Erwinia uredovora, the non-photosynthetic bacterium having the carotenoid synthesis genes can synthesize up to several useful carotenoids such as lycopene, xcex2-carotene, zeaxanthin by using FPP as a substrate. When the genes are combined with the carotenoid synthesis genes of Agrobacterium aurantiacum, the marine bacterium, up to several useful carotenoids such as cantaxanthin and astaxanthin can also be synthesized (See FIGS. 2 and 3). The inventors of the present invention already confirmed that by introducing crt genes of Erwinia uredovora into microorganisms such as Saccharomyces cerevisiae, yeast and Zymomonas mobilis, ethanol-producing bacteria; these microorganisms can produce carotenoids such as xcex2-carotene as anticipated [Yamano, S., Ishii, T., Nakagawa, M., Ikenaga, H., Misawa, N., xe2x80x9cMetabolic engineering for production of xcex2-carotene and lycopene in Saccharomyces cerevisiaexe2x80x9d, Biosci. Biotech, Biochem., 58:1112-1114 (1994); Misawa, N., Yamano, S., Ikenaga, H., xe2x80x9cProduction of xcex2-carotene in Zymomonas mobilis and Agrobacterium tumefaciens by introduction of the biosynthesis genes from Erwinia uredovoraxe2x80x9d, Appl. Environ. Microbiol., 57:1847-1849 (1991); and Japanese laid-open Patent Application No. HEI 3-58786(Japanese Patent Application filing No. HEI 2-53255):xe2x80x9cA DNA chain useful for synthesis of carotenoidsxe2x80x9d by the inventors].
From the above findings, it can be expected that when an appropriate combinations of the carotenoid synthesis genes derived from Er. uredovora and those from marine bacteria(typically the carotenoid synthesis genes derived from Ag. aurantiacum) are introduced into the same microorganism simultaneously, as a principle, all of the microorganisms, in which such genes are introduced and of which introduction-expression system is established, can produce useful carotenoids such as astaxanthin and zeaxanthin.
In such cases, if the IPP isomerase gene(typically, derived from Haematococcus pluvialis, Phaffia rhodozyma and Saccharomyces cerevisiae) is introduced according to the above mentioned method, and is expressed concomitantly with the above carotenoid synthesis gene, higher production amount of useful carotenoids can be achieved.
(4) Deposit of the Microorganisms
The recombinant E. coli strain JM109 has been deposited as follows with the National Institute of Bioscience and Human-Technology, the Agency of Industrial Science and Technology. The strain contains the plasmid having the isolated gene which is the DNA chain of the invention. The names of the plasmids are shown in the parentheses.
(i) JM109(pRH1)
Deposit No.: FERM BP-5032
Date of Receipt: Mar. 6th, 1995
(ii) JM109(pHP11)
Deposit No.: FERM BP-5031
Date of Receipt: Mar. 6th, 1995
(ii) JM109(pSI1)
Deposit No.: FERM BP-5033
Date of Receipt: Mar. 6th, 1995